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article
Mitotic replication initiation proteins are
not required for pre-meiotic S phase
Susan L. Forsburg & Jeffrey A. Hodson
Initiation of mitotic DNA replication in eukaryotes requires conserved factors, including Cdc18/CDC6 and minichromosome maintenance (MCM) proteins. We show here that these proteins are not essential for meiotic DNA replication or subsequent meiotic divisions in fission yeast. In addition, vegetative replication checkpoint genes are not
required for the arrest of meiotic divisions in response to pre-meiotic S-phase delays. Genes essential for other
aspects of vegetative DNA replication, however, including polymerases and DNA ligase, are also required for premeiotic DNA synthesis. Our results indicate that the process of replication initiation and checkpoint control may be
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fundamentally different in mitotic and meiotic cells.
Introduction
Replication origins in eukaryotic cells initiate a single round of
DNA synthesis in each cell cycle.This requires the regulated binding of several conserved initiation proteins, including
CDC6/Cdc18p and the abundant MCM proteins, which form a
‘pre-replicative complex’ (preRC) on the origin. As replication
proceeds, other proteins of the elongation machinery are activated, allowing bulk DNA synthesis1,2. The preRC is inactivated
as replication initiates, ensuring that the origins fire only once
per cell cycle. In fission yeast, many S phase mutants arrest the
cell cycle with apparently replicated DNA, but are unable to enter
M phase. This reflects activation of a checkpoint, and indicates
that replication is actually incomplete or the DNA is damaged3,4.
Thus, we can monitor successful completion of DNA replication
not only by accumulation of bulk DNA, but also by successful
progression through subsequent nuclear division.
DNA also replicates during meiosis. In fission yeast, meiosis
occurs after two haploid cells conjugate to form a transient
diploid resulting in the formation of four haploid spores packaged
in an ascus5. Previous studies of meiotic progression in Schizosaccharomyces pombe have shown that this specialized cell cycle
requires the activity of several general cell cycle regulators that
also function in vegetative cells6–9. Fission yeast also has meiosisspecific genes that may modulate the activity of the global regulators, or may function in independent pathways to control the
onset and progression of the meiotic cell cycle5. Together, these
two sets of genes ensure the production of viable haploid spores.
We characterized the role of vegetative S-phase genes in meiosis
using the same criteria used in vegetative cells. First, we determined
the ability of mutant strains to accumulate replicated DNA. Second,
we assayed their progression through subsequent meiotic divisions.
We expected that the vegetative phenotypes of these mutants would
be analogous to meiotic effects with respect to both these criteria.
We found that mutation of genes involved in initiation had no effect
on meiotic S (meiS) phase or subsequent meiotic divisions. Nevertheless, genes required for progression of DNA replication were also
essential for normal meiotic progression, and showed meiotic phenotypes analogous to those observed in vegetative cells. These
results indicate that the mechanisms coupling DNA replication to
other cell cycle events are different in mitotic and meiotic cells,
whereas the mechanism of bulk DNA synthesis is the same.
Results
Assaying S phase in pat1 mutants
To facilitate the study of genes involved in meiS phase, we used a
temperature-sensitive mutation in the essential Pat1p protein
kinase10,11. If shifted to the restrictive temperature, pat1 mutants
proceed through meiotic DNA replication and both meiotic divisions in the absence of normal meiotic signals. In a haploid, these
meiotic divisions result in uneven segregation of DNA into four
nuclear bodies, and cell death. Nevertheless, this mutant meiosis
depends on the same cell cycle, meiosis-specific and recombination
genes required in normal diploids, making it an accurate and widely
accepted model for the process6,12–14. pat1 thus provides an efficient, synchronous meiosis that allows typical cell cycle analysis.
We arrested pat1 single mutant cells at the G1 of the cell cycle
by nitrogen starvation, and released them to meiosis by re-feeding at the restrictive temperature of 36 °C. The cells completed
meiS phase in four hours (Fig. 1a). By 10 hours, 70%–80% of
cells in a typical experiment completed both meiotic divisions
(Fig. 1e). We combined pat1 with vegetative S phase mutants, the
phenotypes of which have been well characterized. Most of these
mutants were originally isolated because they cause a tight cell
cycle arrest in one cell cycle at 36 °C (ref. 15). These mutants fall
into four broad gene classes: known regulators of the cell
cycle16,17; replication elongation factors18–25; replication initiators26–30; and checkpoint components31–33. Using pat1 to induce
meiosis, we determined the ability of these mutants to complete
meiS phase and subsequent meiotic divisions. We anticipated
that these strains would suffer meiotic defects comparable to
their mutant phenotypes in vegetative cells using both criteria:
synthesis of DNA and completion of nuclear divisions.
In most cases, our assumption was correct (Fig. 1 and Table 1,
and data not shown). Mutations that block enzymes of the replication machinery, or general cell cycle progression, also blocked
meiosis. As observed in studies of cell-cycle regulators, both the
cdc2 kinase and the cdc10 transcription factor mutants arrested
cells before meiS phase, and prevented both meiotic divisions
Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA. Correspondence should be addressed to S.L.F.
(e-mail: [email protected]).
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b
d
e
(Fig. 1c,e, and data not shown6–9). Mutants in the replication
machinery such as cdc22 (ribonucleotide reductase) also blocked
meiS phase and subsequent divisions (Fig. 1c,e), as did pol1
(DNA polymerase-α) and rpa1 (RPA; data not shown). Mutants
of cdc20 (DNA polymerase-ε) underwent a delayed S phase with
a reduction in the efficiency of meiotic divisions (Fig. 1c,e), as did
cdc1 (data not shown). The cdc17 (DNA ligase) mutant completed S phase but reduced the percentage of meiotic divisions
(Fig. 1c,e), as observed for cdc6 (DNA polymerase-δ) and cdc24
(data not shown). These defects were analogous to those seen in
vegetative cells16–25.
The four initiation mutants in our assay, mcm2, mcm4, cdc18
and rad4, showed no obvious meiotic defects. They completed
meiS phase and both meiotic divisions efficiently, with timing
similar to that of the parent strain (Fig. 1d,e, and data not
shown). This contrasts with the mitotic cell cycle, in which all
these genes are known to be involved in replication initiation.
cdc18, mcm2 (cdc19) and mcm4 (cdc21) mutants arrest vegetative
cells with partially replicated DNA and die without dividing29,30,34–36. MCM proteins are also implicated in S-phase progression34, perhaps as a replicative helicase37. rad4 (cut5)
mutants block DNA synthesis entirely, and suffer premature
mitosis from a checkpoint defect26,27. Each of these mutations
tightly arrest vegetative cells in the first cell cycle, and none
undergo normal cell division15,26. Yet none of these mutants had
any discernible effect on meiotic progression, either on DNA
accumulation or on nuclear divisions. Thus, the phenotypes of
these mutants in meiosis and mitosis are quite different.
This difference between these initiation mutants and the other
S phase mutants is unlikely to reflect incomplete thermal inactivation of the mutant proteins. All the initiation mutants tested, and
264
Fig. 1 Meiotic progression in pat1
mutant strains. a, meiS phase measured by flow cytometry in pat1-114
(FY800) mutants is completed in 4 h.
Completion of meiS phase was scored
as the conversion of cells with a 1C
DNA content to 2C. b, Recombination
mutants pat1-114 ∆rec12 (FY966) and
pat1-114 ∆rec12 mcm4/cdc21-M68
(FY1123) complete meiotic replication. c, Mutants affecting global cell
cycle
regulators
or
replication
enzymes show absent or delayed S
phase: pat1-114 cdc2-M26 (FY770),
pat1-114 cdc22-M45 (FY576), pat1-114
cdc17-K42 (FY585), pat1-114 cdc20M10 (FY624). d, Initiation mutants
complete S phase with timing similar
to that of pat1-114 alone: pat1-114
mcm2/cdc19-P1 (FY574), pat1-114
mcm4/cdc21-M68 (FY623), pat1-114
rad4-116 (FY644), pat1-114 cdc18-K46
(FY773). e, Completion of the MII division at 10 h was determined by counting the percentage of cells with ≥3
nuclei (because of irregular segregation, the fourth nucleus is not always
apparent in a pat1 mutant). No
mutants completed MI without completing MII (data not shown).
only those, were proficient at
meiosis in a pat1 background. It
is doubtful that only the initiation mutants would be leaky,
and only in this process, especially because they show no
delays in timing relative to the
parent strain. Nevertheless, we
repeated the assay using a
method that did not rely on temperature-sensitive alleles. We constructed a ∆cdc18 pat1 strain in which the cells were kept alive by
an integrated copy of cdc18+ under control of a regulated promoter. Shutting off cdc18+ expression by the addition of thiamine
results in a tight null phenotype in one mitotic cell cycle, in which
initiation and DNA synthesis are blocked30,38. In meiosis, this
mutant proceeded efficiently through meiS phase and meiotic
divisions whether the promoter was induced or repressed (Fig. 2).
Thus, the meiotic phenotype we observed does not represent
incomplete thermal inactivation of the gene products.
Because the replication initiation proteins are not essential for
meiosis, we asked whether they are actually expressed. pat1
mutant cells undergo meiosis-specific gene expression upon
shift to restrictive temperature14,39. We characterized the concentrations of MCMs and other replication proteins in pat1
induced meiosis (Fig. 3a). Of the proteins we examined, we
detected only PCNA in starved pat1 cells. Concentrations of
other proteins rose by the time of meiS phase, including the
MCM proteins, Orp1 and p34cdc2. Tyrosine phosphorylation of
p34cdc2 was apparent at this time, but was lost around the time of
the second meiotic division, agreeing with previous observations40 (Fig. 3a, and data not shown). We compared the protein
concentrations in pat1 mcm4 mutants (Fig. 3b). Although the
kinetics of meiosis are similar in both strains (Fig. 1, and data
not shown), we did not detect the Mcm4 protein, and observed
reduced concentrations of Mcm2p in the pat1 mcm4 strain. Nevertheless, p34cdc2 accumulated and tyrosine phosphorylation of
p34cdc2 rose and fell with similar timing as in the mcm4+ strain.
When the analogous experiment was performed in vegetative
cells, and mcm4 mutants were released from starvation under
conditions in which concentrations of temperature-sensitive
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Table 1 • pat1 assay results
Classification
cell-cycle regulator
replication machinery
initiation
checkpoint
Cds1/RAD53 checkpoint kinase32
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recombination
Mutant allele
Product
cdc2-M26
cdc10-V50
cdc22-M45
pol1-1
rpa1/rad11A
cdc20-M10
cdc1-7
cdc17-K42
cdc6-23
cdc24-M38
mcm2/cdc19-P1
mcm4/cdc21-M68
cdc18-K46
rad4-116
∆cds1:ura4+
CDK17
transcription factor16
ribonucleotide reductase23
catalytic subunit, DNA polymerase-α24
RPA25
catalytic subunit, DNA polymerase-ε20
subunit, DNA polymerase-δ21
DNA ligase18
catalytic subunit, DNA polymerase-δ22
novel19
MCM228
MCM429
CDC6 homologue30
DPB11 homologue26,27
∆chk1:ura4+
∆rad3:ura4+
∆rec12:LEU2
∆rec12:LEU2 mcm4/cdc21-M68
CHK1 checkpoint kinase31
ATM-like checkpoint kinase33
SPO11 homologue14,42
Mcm4p is similarly undetectable, the cells failed to replicate
their DNA and suffered irreversible cell cycle defects34.
Thus, although the proteins are apparently expressed in meiotic cells, there is no obvious effect in their absence. They may be
unnecessary, they may have non-essential meiotic functions, or
they may overlap with a meiosis-specific redundant pathway.
Recombination genes do not substitute for MCM
function
2C DNA
content
no
no
no
no
no
slow
slow
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
Completion of
meiotic divisions
no
no
no
no
no
no
no
no
no
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
tested known vegetative checkpoint mutants using hydroxyurea
(HU) to block replication, or the cdc17 mutant to induce damage,
in the same pat1 background used previously. Cds1p and Chk1p
kinases act in parallel, responding to HU or DNA damage, respectively. Rad3p kinase is upstream, and is required for all DNA metabolism checkpoints3,4. Treatment with 15 mM HU blocked S-phase
progression (data not shown), and also reduced efficiency of both
meiotic divisions in pat1 single mutants. We observed no increase
in the percentage of meiotic divisions in the double mutants (Fig.
4). We tested the response of ∆chk1 mutants to DNA damage in a
cdc17 ∆chk1 pat1 triple mutant; again, this strain did not show any
obvious increase in meiotic divisions over the cdc17 pat1 mutant
alone (Fig. 4). Thus, the cells were apparently able to maintain their
arrest even in the absence of the vegetative checkpoint proteins. As
described in previous sections, there is no obligate dependency of
meiotic progression on recombination. Because the mutants were
arrested in S phase, we could not determine the effect of these
checkpoint challenges on recombination. Thus, although meiotic
divisions are reduced in the presence of replication arrest, this
reduction seems to be independent of vegetative checkpoint genes,
suggesting there is a meiosis-specific replication checkpoint or
dependency mechanism.
In some situations, replication can be initiated by recombination41.
To test whether a meiotic recombination mechanism substitutes for
the normal initiation proteins in meiosis, we inactivated an early
step in meiotic recombination using the ∆rec12 mutant14 and asked
whether the cells now required mitotic initiation factors. rec12+
encodes the fission yeast homologue of budding yeast SPO11,
which is required for the formation of double-stranded breaks42.
Both the pat1 ∆rec12 mcm4 mutant and the pat1 ∆rec12 mutant
accumulated replicated DNA and proceeded through subsequent
meiotic divisions (Fig. 1b,e). There was a modest, but reproducible,
reduction in the kinetics of S phase and meiotic divisions. On the
other hand, as the cells successfully completed replication and meiotic divisions, we conclude that there is no direct redundancy
between MCM function and Rec12-mediated recombination in
initiating meiS phase. The ability of the rec12 mutant to complete Meiosis in diploids shows equivalent results
meiosis also indicates that completion of meiotic divisions does not We constructed diploid strains homozygous for the mutations
depend on successful entry into recombination.
under study and assayed the completion of meiosis at both
Checkpoint genes are not required to restrain
meiotic divisions
In vegetative cells, mutants that arrest late in S phase with a 2C DNA
content do not proceed into mitosis because of an active checkpoint3,4. Thus, another possible explanation for the lack of phenotype associated with the initiation mutants is that they are actually
defective in S phase, but lack a checkpoint to restrain their subsequent meiotic divisions (Fig. 1 and Table 1); however, this is
unlikely. First, all the S-phase elongation mutants we tested were
unable to carry out meiotic divisions. This suggests an intact checkpoint mechanism that prevents meiotic division when S phase is
incomplete, just as in vegetative cells. Second, in vegetative cells,
mcm mutants engage the same checkpoints as other S-phase
mutants34,36. We investigated this putative meiotic checkpoint
more closely, predicting that any mutation abrogating a meiotic
replication checkpoint should be unable to restrain meiotic divisions in the presence of damage or arrested DNA replication. We
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a
b
Fig. 2 Meiotic and mitotic progression in a strain where cdc18+ expression is
shut off by the addition of thiamine. a, meiS phase measured by flow cytometry in pat1-114 ∆cdc18:[REP41-cdc18+] (FY589) in the presence or absence of
thiamine was completed by 8 h. Completion of meiS phase was scored as the
conversion of cells with a 1C DNA content to 2C. b, Completion of the MII division at 8 h was determined by counting the percentage of cells with ≥3 nuclei.
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a
Fig. 3 Protein concentrations in pat1 mutant cells, synchronized as described in
Fig. 1. a, pat1 mutant cells. Two strains, FY903 (pat1 mcm2HA) and FY800 (pat1
orp1HA), were used to allow monitoring of two different HA-tagged proteins.
Kinetics of protein accumulation and meiotic progression were identical (Fig. 1,
data not shown). b, pat1 mcm4 mutant cells (FY955: pat1 mcm4 mcm2HA).
Lane A, asynchronous vegetative cells.
with normal segregation of markers (data not shown). In contrast, the ∆rec12/∆rec12 spores had severely reduced viability,
consistent with their abnormal morphology. Of the few ∆rec12
survivors, many were diploid, and segregation of markers was
abnormal. Thus, the meiotic products formed in the initiation
mutants were indistinguishable from wild-type meiotic products, in contrast to the recombination mutant. This indicates that
these initiation mutants do not affect normal diploid meiosis.
Discussion
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b
restrictive (34 °C) and permissive (25 °C) temperatures. No pat1
mutation was present in these strains. This allowed us to verify
that the results in the pat1 mutants are consistent with events in
normal meiS phase. Moreover, we were able to monitor spore
viability. We reasoned that if the meiotic divisions in the initiation mutants resulted from a defect in a meiosis-specific checkpoint, the resulting spores should show reduced viability relative
to wild type. On the other hand, if they are normal meiotic divisions, viability should be maintained. The temperature-sensitive
diploids showed a cdc phenotype and were unable to form vegetative colonies at 34 °C (data not shown), which is the maximum
permissive temperature for meiosis in wild-type S. pombe and is
fully restrictive for the haploid mutants (data not shown).
In all cases the results with diploids agreed with the pat1 assay,
indicating that the initiation mutants were not essential for meiosis (Fig. 5a). rad4/rad4, mcm2/mcm2, mcm4/mcm4, cdc18/cdc18
and ∆rad3/∆rad3 diploids sporulated efficiently even at the
restrictive temperature, whereas other replication mutants were
severely defective. We characterized the morphology of the asci
produced in meiosis at 34 °C. Asci from wild-type, rad4/rad4,
mcm4/mcm4 or ∆rad3/∆rad3 diploids were all normal in size,
forming four normal spores with equivalent amounts of 4,6diamidino-2-phenylindole (DAPI) staining material in each (Fig.
5b). In contrast, few asci were produced in the cdc17/cdc17
diploid, and they were very irregular in size, number of spores and
distribution of DNA. ∆rec12/∆rec12 and ∆rec12 mcm4/∆rec12
mcm4 also completed meiosis efficiently, although with many
abnormal asci, a high proportion of dyads and binucleate spores,
and unequal segregation of DAPI staining, as expected for strains
with a severe recombination defect14.
We took asci from cultures incubated at 25 °C or 34 °C and dissected tetrads at 25 °C to assess the viability of the meiotic products (Fig. 5c). Wild-type, rad4/rad4 and mcm4/mcm4 asci
produced at either temperature formed four viable spore clones,
266
Our results indicate that replication initiation proteins of the
mitotic pre-RC are not essential for meiS phase, using two criteria: ability to synthesize DNA and ability to complete both meiotic divisions. We also determined that checkpoint mutants do
not affect the response to damage or HU treatment. On the other
hand, genes defining elements of the replication machinery are
conserved between both S phases; in all cases, meiotic divisions
were blocked, even when DNA accumulated. This may be true for
other systems as well. The S. cerevisiae CDC7 gene is required for
meiotic recombination but not for meiS phase43,44. CDC7
encodes an essential MCM-kinase that is required to fire individual origins of replication in vegetatively growing cells45. If the
role of CDC7 is to activate the replication origins by modifying
the MCM proteins, and CDC7 is not required for meiotic replication, then we predict that the MCMs will also be dispensable
for budding yeast meiS phase. We have not yet examined whether
the initiation mutants affect recombination frequencies, but they
suffer no obvious segregation defects.
Meiotic S phase in most species is much slower than that in
vegetative cells, which could indicate a reduced number of origins firing, more staggered origin firing, or less processive elongation46–48. It remains a formal possibility that the difference
between vegetative and meiotic S phase is not qualitative, but
quantitative: reduced concentrations of initiation proteins may
Fig. 4 Vegetative checkpoint mutants do not affect meiotic checkpoint arrest.
Strains pat1-114 (FY903), pat1-114 ∆cds1 (FY900), pat1-114 ∆chk1 (FY596),
pat1-114 ∆rad3 (FY821), pat1-114 ∆chk1 cdc17 (FY595), pat1-114 ∆cdc17
(FY585) were cultured as described in Fig. 1; upon release to meiosis, the indicated cultures received hydroxyurea (HU) to 15 mM final concentration to
block replication. DNA replication was monitored by FACS to verify that HU
treatment blocked meiotic S phase (data not shown). pat1-114 ∆cds1 ∆chk1
behaved identically to pat1-114 ∆rad3 (data not shown).
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b
article
c
Fig. 5 Effects of S-phase mutants in homozygous diploids. a, Ratio of cells
completing meiosis at the restrictive temperature (34 °C) compared with the
permissive temperature (25 °C). A value of 1.0 indicates equally efficient formation of asci at both temperatures. b, Photomicrographs of DAPI-stained
diploids following incubation at the restrictive temperature shows normal
four-spored assay in initiation mutants. Bar, 10 µm. c, Viability of spores produced during meiosis at 34 °C (black boxes) or 25 °C (hatched boxes), determined by tetrad dissection at 25 °C, and calculated as percentage spores
formed per theoretical maximum of four spores/tetrad. ND, not done, as a
null allele is not temperature dependent.
cedes recombination, a period of genome plasticity that may
impose further requirements on the replication process. It is perhaps not surprising that different proteins are required for the
orderly execution of meiotic events in response to these unique
signals. Fission yeast provides us a powerful genetic system to
identify meiosis-specific replication factors, and we are carrying
out a genetic screen for such proteins.
Methods
Yeast strains and culture. All strains are congenic to 972 h- and cultured
using standard methods. We constructed double-mutant strains by tetrad
dissection (for temperature-sensitive mutants) or by random spore analysis (for disruption mutants).
be sufficient for meiotic progression if fewer replication origins
fire. Nevertheless, we consider this unlikely. Work in budding
yeast has shown that the same replication origins are used in
mitotic and meiotic cells44,49. In the case of the fission yeast
MCM proteins, we know that even modest reduction in their
vegetative concentrations results in severe defects including
genetic instability, delayed DNA replication and cell death,
although bulk DNA synthesis can still occur34. This is consistent
with a role for MCMs during S-phase progression, and agrees
with their implied role as a replicative helicase37. In contrast, in
meiosis we showed that the mcm mutants not only replicate their
DNA, but efficiently complete both meiotic divisions to produce
viable spores with normal timing, despite undetectable concentrations of MCM protein. This is not simply an MCM-related
observation: the initiation mutants cdc18 and rad4 behave similarly, although their vegetative phenotypes are quite different. We
conclude that the requirements for all these initiation factors are
significantly different in meiosis and mitosis.
Our results suggest that mechanisms coupling meiS phase to
upstream and downstream events in meiosis are distinct from
mechanisms operating in mitotic cells. Meiosis is not a cycle, but
a terminal differentiation pathway, which occurs under specific
environmental conditions5. Meiotic DNA replication also prenature genetics • volume 25 • july 2000
Meiosis assays. For pat1 assays, we grew strains to late exponential phase,
washed the strains into nitrogen-starvation medium (EMM-N) containing
7 µg/ml adenine, and incubated them overnight at 25 °C to arrest most cells
in G1 phase of the cell cycle (1C DNA content). Percentage of G1 arrest varied in different mutant backgrounds. We shifted cultures to 36 °C and refed them with an equal amount of pre-warmed medium containing 1 g/l of
NH4Cl and 70 µg/ml uracil, adenine and leucine. We collected aliquots
every hour and fixed them for fluorescence-activated cell sorting (FACS);
we stained cells with Sytox Green or propidium iodide as described34. We
used the shift of the 1C peak to the 2C position to indicate meiS phase. We
stained samples with DAPI and the nuclei were counted under fluorescence
microscopy with 1, 2 or ≥3 nuclei indicating no divisions, MI division or
MII division, respectively. For simplicity, only MII division data are shown;
no mutants completed MI without completing MII. Representative experiments are presented. We repeated time courses at least three times for each
strain, and each experiment included a pat1 mutant as a control. In the
indicated experiments, we treated cells with 15 mM HU or 15 µM thiamine
at the time of re-feeding.
For diploid assays, we constructed fresh homozygous strains carrying
the indicated mutations by crossing strains with complementing ade6
markers: wild type (FY254/FY261); ∆rec12/∆rec12 (FY1012/FY1013);
∆rec12
mcm4/∆rec12
mcm4
(FY1145/FY1146);
∆rad3/∆rad3
(FY1106/FY1107);
cdc18/cdc18
(FY919/FY922);
mcm2/mcm2
(FY243/F459); mcm4/mcm4 (FY784/FY786); rad4/rad4 (FY1113/FY1114);
cdc17/cdc17 (FY569/FY969); cdc22/cdc22 (FY331/FY583); cdc24/cdc24
(FY654/FY655); pol1/pol1 (FY1109/1110); ∆rad3/∆rad3 (FY1106/FY1107).
We grew cultures to mid-exponential phase and starved them overnight in
EMM medium lacking nitrogen and glucose, but containing 7 µg/ml uracil
and leucine, then inoculated them to a final concentration of 0.2% glucose
and 1% glycerol to release into meiosis at permissive (25 °C) or restrictive
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(34 °C) temperatures as described50. None of the temperature-sensitive
diploid strains were capable of colony formation at 34 °C (data not shown),
the maximum permissive temperature allowing spore formation in wildtype cells. Following 24–36-h incubation, we examined cultures microscopically to determine the percentage of asci produced at both
temperatures. Under these conditions, the release of the mutants to meiosis
was not sufficiently synchronous to allow meaningful FACS, therefore successful completion of S phase was inferred by the formation of spores and
generation of viable meiotic products with normal marker segregation. We
repeated time courses at least three times for each strain; representative
data are shown. We stained samples with DAPI and photographed them
under fluorescence using a Leitz microscope and SPOT-2 CCD camera system. We dissected tetrads produced by the indicated strains following incubation at 25 °C or 34 °C onto YE plates at 25 °C. We determined viability by
counting the total number of viable colonies produced from each ascus,
assuming a theoretical maximum of four viable colonies per dissected
tetrad. Thus, only 4 viable spore clones/tetrad would yield viability of
100%.
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Protein analysis. We sampled aliquots during exponential growth (lane A,
asynchronously growing vegetative cells). Following starvation and release to
meiosis, we took samples hourly (0– 8) and monitored for DNA content and
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Acknowledgements
We thank T. Carr, B. Grallert, O. Nielsen, N. Rhind, C. Shimoda and G.
Smith for strains; B. Grallert, G. Karpen and M. McKeown for discussions;
and T. Hunter, G. Karpen, M. McKeown, S. Pasion and T. Pollard for critical
reading of the manuscript. S.L.F. acknowledges a guest professorship from the
University of Copenhagen and the hospitality of O. Nielsen and R. Egel in
developmental stages of this project. This work was supported by National
Institutes of Health grant GM54797 to S.L.F., who is a scholar of the
Leukemia & Lymphoma Society.
Received 3 December 1999; accepted 4 April 2000.
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nature genetics • volume 25 • july 2000